Résumé
Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.
Sujets)
Animaux , Lapins , Cholestérol/métabolisme , Érythrocytes/métabolisme , Hémolysines/pharmacologie , Passiflora/composition chimique , Saponines/pharmacologie , Chromatographie en phase liquide à haute performance , Érythrocytes/effets des médicaments et des substances chimiques , Hémolyse , Hémolysines/isolement et purification , Double couche lipidique/métabolisme , Extraits de plantes/pharmacologie , Feuilles de plante/composition chimique , Saponines/isolement et purificationRésumé
Porin isolated from bovine skeletal muscle was reconstituted in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 + 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +- 0.08 nm.
Sujets)
Animaux , Techniques in vitro , Canaux ioniques/physiologie , Double couche lipidique , Mammifères/physiologie , Porines , Techniques de patch-clampRésumé
The effective size of colicin Ia channel was tested by a recently described method 9FEMS, Microbiology and Immunology (1992). 105: 93-100) in which the nonelectrolyte molecules with different hydrodynamic diameters (0.52 to 5.0nm) were used as molecular tools. We have shown that despite low conductance (55-105 pS at 1.5 MKCl, pH 7.0) the ion channels formed by colicin Ia have a fairly large water pore diameter equal to 1.66-1 1.88nm. The results are discussed in terms of an energetic barrier for ions passing into the channel lumen